Influence of serum proteins on conformation of prostate-specific antigen.

Partition behavior of prostate-specific antigen (PSA) was studied in aqueous Dextran-Ficoll two-phase system. It was found that the partitioning of PSA changed in the presence of other proteins, in particular, bovine serum albumin, human serum albumin, human transferrin, and human gamma-globulin. The partition coefficient of PSA in mixtures with increasing amounts of these proteins decreased along the S-shaped curve and dropped to essentially the same value at the 10(4)-10(5) protein: PSA molar ratio. Partition behavior of the above proteins was examined separately. Partition coefficient of a protein represents the protein solvent exposed residues; i.e., it reflects the 3D-structure of the protein in solution. Partition of binary protein mixtures reflects the interaction of the two proteins and therefore characterizes the PSA-induced conformational changes in a protein agent and the change in the PSA conformation induced by a protein agent. In other words, the protein effect on the partition behavior of free PSA may be explained by the effect of the non-specific PSA-protein interactions on PSA conformation. Formation of such PSA-protein encounter complexes was shown to be dominated by the electrostatic forces, since the efficiency of a given protein-agent to induce changes in the partition behavior of PSA was proportional to its absolute mean net charge. Furthermore, in agreement with the earlier hypothesis that the protein segments with increased dynamic propensities (i.e., 'discrete breathers') can be important for conformational transitions accompanying binding processes, our analysis of intrinsically disordered regions (IDR) in all the proteins examined showed that the propensity for intrinsic disorder is related to the PSA partition-modulating capability of the protein.

Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini.

The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO3-:Na+ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser982. pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS1026), and an additional site in its amino-terminus (KRKT49). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser1026 following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser1026 with Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (GDS) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr49, which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in GDS. In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO3-:Na+ stoichiometry of 2:1, 8-Br-cAMP increased GDS by ~90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser1026 mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr49 plays an essential role in the cAMP-induced increase in GDS.